A multilayer microfluidic chip integrating nucleic acid extraction and purification with digital detection

Abstract

The present invention relates to a multilayer microfluidic chip integrating nucleic acid extraction and purification with digital detection, wherein the multilayer microfluidic chip comprises, from top to bottom, a PDMS-based nucleic acid extraction and purification layer (100), a glass substrate (200) and a silicon-based microcavity digital PCR detection layer (300). The present invention integrates sample preparation and digital PCR detection, and can be applied to the precise quantitative detection of pathogens; compared with the existing pathogen detection platform, it has great advantages in detection sensitivity, process automation, integration, etc., and has broad application prospects in molecular diagnosis.

Description

Technical Field

[0001] The present invention belongs to the field of pathogen detection platforms, and in particular relates to a multilayer microfluidic chip integrating nucleic acid extraction and purification with digital detection. Background Art

[0002] Nucleic acid detection technology is the most widely used molecular diagnostic technology in recent years. In the diagnosis of infectious diseases, this technology can quickly and efficiently amplify the characteristic nucleotide sequences of pathogens, and has been widely used in clinical diagnosis, disease screening and other fields. Traditional nucleic acid detection steps usually include: sample inactivation, nucleic acid extraction and purification, nucleic acid amplification and detection. Among them, nucleic acid extraction and purification are the key in molecular biology experiments. It is related to whether high-quality nucleic acids can be extracted, which can directly affect the success or failure of subsequent experiments. For nucleic acid extraction and purification, the reagents provided by the current test kits often have cumbersome steps, take a long time, and are prone to cross-contamination due to the operation method or experimental environment, so there are high requirements for the experimental ability of medical personnel and the testing environment.

[0003] Nucleic acid amplification and detection is to efficiently and quickly amplify the characteristic nucleotide sequences of the extracted and purified pathogens and perform fluorescent or visual detection to obtain immediate diagnostic results. The duration, sensitivity and specificity of the nucleic acid amplification method will also directly affect the interpretation of subsequent results. At present, the commonly used method for nucleic acid amplification detection is real-time fluorescent quantitative PCR, which can perform qualitative and quantitative analysis of nucleic acids in samples and is the current gold standard for nucleic acid amplification detection. However, its disadvantage is that the quantitative analysis of this method must rely on the existence of a standard curve, and it is impossible to achieve absolute quantification of nucleic acid molecules. In addition, when the nucleic acid content is low, it is impossible to detect low-abundance target genes, resulting in false negative results.

[0004] In response to the above phenomenon, some studies have integrated nucleic acid extraction and purification with more sensitive digital detection in the chip, and simplified the detection process based on magnetic bead transfer or driven reagent flow distribution. The shortcomings of current detection methods are often that integrated detection is difficult and the operation is cumbersome, often requiring the use of negative pressure or centrifugation to drive liquid flow. Therefore, the application of highly sensitive and easy-to-operate devices that integrate nucleic acid extraction and purification with digital detection in the field of molecular diagnosis still has problems that need to be solved. Summary of the invention

[0005] The technical problem to be solved by the present invention is that the prior art has the problem of difficult integrated detection, complicated operation and the need to use negative pressure or centrifugal drive to drive the liquid flow.

[0006] The present invention provides a multi-layer microfluidic chip integrating nucleic acid extraction and purification with digital detection, wherein the multi-layer microfluidic chip comprises a PDMS-based nucleic acid extraction and purification layer, a glass substrate and a silicon-based microcavity digital PCR detection layer from top to bottom; wherein the PDMS-based nucleic acid extraction and purification layer is composed of a liquid storage chamber, a bottom connecting pipe, a microcolumn array, a capillary burst valve, a microcavity detection layer inlet connection hole and a microcavity detection layer outlet connection hole; the liquid storage chamber comprises a lysate storage chamber, a first washing liquid storage chamber, a second washing liquid storage chamber, a mineral oil storage chamber and an eluent storage chamber connected in sequence through a bottom connecting pipe; the microcolumn array is used for water-oil phase separation of the mineral oil storage chamber and the eluent storage chamber, and the capillary burst valve is used to prevent the liquid in the eluent storage chamber from spontaneously entering the inlet connection hole during the nucleic acid purification process. Only when the oil phase is injected into the mineral oil storage chamber and pressure is applied, the water phase will break through the capillary burst valve and enter the silicon-based microcavity digital PCR detection layer through the inlet connection hole.

[0007] The lysing liquid storage chamber, the first washing liquid storage chamber and the second washing liquid storage chamber utilize the water-in-oil principle to achieve non-interference between the lysing liquid, the first washing liquid and the second washing liquid.

[0008] The glass substrate is provided with openings corresponding to the microcavity detection layer sample inlet connection hole and the microcavity detection layer sample outlet connection hole.

[0009] The silicon-based microcavity digital PCR detection layer comprises a microcavity inlet, a flow channel, a microporous reaction chamber and a sample outlet; the microcavity inlet is connected to the microcavity detection layer inlet connection hole, and the sample outlet is connected to the microcavity detection layer outlet connection hole; the microporous reaction chamber is connected to the microcavity inlet and the sample outlet through the flow channel.

[0010] The eluent storage chamber is provided with an eluent injection hole.

[0011] The microporous reaction chamber is composed of a plurality of micropores distributed in the flow channel, and the spacing and size of the micropores are kept consistent, so as to facilitate the digital analysis of subsequent detection.

[0012] The present invention also provides a method for using a multilayer microfluidic chip integrating nucleic acid extraction and purification with digital detection, comprising the following steps:

[0013] (1) First, an aqueous elution reagent (aqueous phase) is pre-filled into the elution liquid reservoir chamber, and then mineral oil is injected into the lysis liquid reservoir chamber and the chip is tilted so that the mineral oil flows through the bottom connecting pipe through the first washing liquid reservoir chamber, the second washing liquid reservoir chamber, and the mineral oil reservoir chamber and fills the chamber; then, a nucleic acid extraction and purification reagent is injected into the corresponding reservoir chamber, and each reagent is separated in an independent chamber;

[0014] (2) During the detection process, the sample is added to the lysis solution storage chamber, the cells or viruses in the sample are lysed, and the released nucleic acid molecules are adsorbed by the nucleic acid extraction magnetic beads, and then the nucleic acid extraction magnetic beads are dragged into the subsequent reaction chamber to complete the washing and elution process to obtain the PCR reaction solution;

[0015] (3) The other liquid storage chambers except the mineral oil liquid storage chamber are sealed with adhesive tape, and pressure is applied by continuously injecting the oil phase into the mineral oil liquid storage chamber. The oil phase pushes the water phase into the silicon-based microcavity digital PCR detection layer in an orderly manner through the microcavity detection layer injection port connection hole; finally, the chip is placed in an in-situ PCR instrument and the relevant programs are set. After the reaction is completed, the detection results are observed under a fluorescence microscope.

[0016] Principle of the invention:

[0017] The present invention integrates nucleic acid extraction and purification with digital PCR detection. The nucleic acid extraction and purification part is designed based on the immiscible interface of water and oil surface tension. The chip separates the nucleic acid extraction and purification reagents into reagent droplets in a separate chamber through the oil phase or maintains the water-oil interface through a microcolumn array so that it exists independently and stably in the chamber, ensuring that each reagent reacts independently during the operation without interfering with each other. The transfer of nucleic acid molecules is completed by dragging magnetic beads through the bottom connecting channel to shuttle in each reagent droplet to complete the adsorption and enrichment, washing and elution of nucleic acid molecules. The digital PCR detection part is designed based on the sample preparation of the nucleic acid extraction and purification part, and accurate absolute quantitative detection can be achieved through limiting dilution and Poisson distribution statistics.

[0018] Beneficial Effects

[0019] The multilayer microfluidic chip of the present invention integrates sample preparation and digital PCR detection, and can be applied to the precise quantitative detection of pathogens. The specially designed water-oil separation structure (microcolumn array) of the present invention not only has the function of reagent separation in the nucleic acid extraction and purification part, but also only needs to continuously inject the oil phase into the oil cavity to provide pressure during the subsequent microcavity digital detection, so that water and oil can enter the microcavity in an orderly manner and fill it. No complex equipment such as a negative pressure pump or a centrifuge is required to drive the liquid flow, so as to achieve simple semi-automatic sampling. The present invention allows the target sample to be sent into the PCR instrument to complete the detection of the target sample by simply injecting reagents and operating the magnet during the detection process, which greatly reduces the requirements for the professional skills and auxiliary equipment of the operator, and at the same time makes the present invention potentially applicable in the field of molecular diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the structure of the multilayer microfluidic chip of the present invention;

[0021] Figure 2 This is a schematic diagram of the structure of the PDMS-based nucleic acid extraction and purification layer in the multi-layer microfluidic chip of the present invention;

[0022] Figure 3 This is a schematic diagram of the structure of the silicon-based microcavity digital PCR detection layer in the multi-layer microfluidic chip of the present invention;

[0023] Figure 4 is a cross-sectional view of the multilayer microfluidic chip of the present invention;

[0024] Figure 5 Schematic diagram of the use process of the multilayer microfluidic chip of the present invention;

[0025] Figure 6 The ORF1ab gene purification and digital detection results of SARS-CoV-2 were performed using the multilayer microfluidic chip of the present invention;

[0026] Among them, (A) concentration 10 5 cp / μL, (B) concentration 10 3 cp / μL, (C) concentration 10 1 cp / μL, (D) blank.

[0027] Figure 1 to Figure 5 The accompanying drawings are denoted as follows:

[0028] 100 is a PDMS-based nucleic acid extraction and purification layer;

[0029] 101 is a bottom connecting pipe;

[0030] 102 is a micro-pillar array;

[0031] 103 is an eluent injection hole;

[0032] 104 is a capillary burst valve;

[0033] 105 is a microcavity detection layer injection port connection hole;

[0034] 106 is a sample outlet connection hole of the microcavity detection layer;

[0035] 110 is a liquid storage chamber;

[0036] 111 is a lysate storage chamber;

[0037] 112 is a first washing liquid storage chamber;

[0038] 113 is a second washing liquid storage chamber;

[0039] 114 is a mineral oil storage chamber;

[0040] 115 is an eluent storage chamber;

[0041] 120 is a reaction reagent;

[0042] 121 is a lysis reagent;

[0043] 122 is a first washing liquid;

[0044] 123 is a second washing liquid;

[0045] 124 is mineral oil;

[0046] 125 is an elution (amplification) reagent;

[0047] 131 is a nucleic acid extraction magnetic bead;

[0048] 200 is a glass substrate;

[0049] 300 is a silicon-based microcavity digital PCR detection layer;

[0050] 301 is a microcavity injection port;

[0051] 302 is a flow channel;

[0052] 303 is a microporous reaction chamber;

[0053] 304 is a sample outlet. DETAILED DESCRIPTION

[0054] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0055] Example 1

[0056] Depend on Figure 1-Figure 2 , Figure 4As shown, this embodiment provides a multi-layer microfluidic chip integrating nucleic acid extraction and purification with digital detection, wherein the multi-layer microfluidic chip comprises, from top to bottom, a PDMS-based nucleic acid extraction and purification layer 100, a glass substrate 200, and a silicon-based microcavity digital PCR detection layer 300; wherein the PDMS-based nucleic acid extraction and purification layer 100 comprises a liquid storage chamber 110, a bottom connecting pipe 101, a microcolumn array 102, a capillary burst valve 104, a microcavity detection layer sample inlet connection hole 105, and a microcavity detection layer sample outlet connection hole 106; the liquid storage chamber 110 comprises a plurality of connecting holes 104, 105, 106, 107, 108, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 1 The lysis liquid storage chamber 111, the first washing liquid storage chamber 112, the second washing liquid storage chamber 113, the mineral oil storage chamber 114 and the eluent storage chamber 115; the microcolumn array 102 is used for water-oil phase separation of the mineral oil storage chamber 114 and the eluent storage chamber 115, and the capillary burst valve 104 is used to prevent the liquid in the eluent storage chamber 115 from spontaneously entering the injection port connecting hole 105 during the nucleic acid purification process. Only when the oil phase is injected into the mineral oil storage chamber 114 and pressure is applied, the water phase will break through the capillary burst valve 104 and enter the silicon-based microcavity digital PCR detection layer through the injection port connecting hole 105.

[0057] The lysing liquid storage chamber 111, the first washing liquid storage chamber 112, and the second washing liquid storage chamber 113 use the water-in-oil principle to ensure that the lysing liquid, the first washing liquid, and the second washing liquid do not interfere with each other.

[0058] The glass substrate 200 is provided with openings corresponding to the microcavity detection layer sample inlet connection hole 105 and the microcavity detection layer sample outlet connection hole 106 .

[0059] Depend on Figure 3 As shown, the silicon-based microcavity digital PCR detection layer 300 includes a microcavity inlet 301, a flow channel 302, a microporous reaction chamber 303 and a sample outlet 304; the microcavity inlet 301 is connected to the microcavity detection layer inlet connection hole 105, and the sample outlet 304 is connected to the microcavity detection layer sample outlet connection hole 106; the microporous reaction chamber 303 is connected to the microcavity inlet 301 and the sample outlet 304 through the flow channel 302.

[0060] The eluent storage chamber (115) is provided with an eluent injection hole (103).

[0061] Depend on Figure 5 As shown, this embodiment also provides a method for using a multilayer microfluidic chip integrating nucleic acid extraction and purification with digital detection, including:

[0062] (1) Detection preparation stage: First, 6 μl of aqueous elution reagent 125 (aqueous phase) is injected into the eluent storage chamber 115 from the eluent injection hole 103, and then mineral oil 124 (oil phase) is injected into the lysate storage chamber 111 and the chip is tilted so that the mineral oil flows through the bottom connecting pipe 101 through the first washing liquid storage chamber 112, the second washing liquid storage chamber 113, and the mineral oil storage chamber 114 and fills them. A sufficient amount of mineral oil should be maintained in each storage chamber. The reagents used for nucleic acid extraction and purification are nucleic acid lysate 121, first washing liquid 122, and second washing liquid 123, respectively. The above reagents are injected into the lysate storage chamber 111, the first washing liquid storage chamber 112, and the second washing liquid storage chamber 113 in sequence, and the injected reagent volumes are 8 μl, 4 μl, and 4 μl, respectively.

[0063] (2) Sampling: Take 2 μl of sample and inject it into the lysis solution storage chamber 111.

[0064] (3) Nucleic acid extraction and purification: Nucleic acid lysis solution 121, first washing solution 122, and second washing solution 123 form reagent droplets in mineral oil 124. Elution reagent 125 and mineral oil 124 maintain a stable water-oil interface through micro-pillar array 102 and are stably stored in eluent storage chamber 115 through capillary burst valve 104. Under the action of magnet, nucleic acid extraction magnetic beads 131 move longitudinally to mix lysis solution reagent 121, and move transversely to transfer nucleic acid molecules to first washing solution 122, second washing solution 123 in sequence.

[0065] 3. Mineral oil 124 and elution reagent 125. The nucleic acid molecules are released in the mixture of the lysate reagent 121 and the sample and combined with the nucleic acid extraction magnetic beads 131. The residual impurities are washed in the first washing solution 122 and the second washing solution 123. The nucleic acid molecules carried by the magnetic beads are eluted in the elution reagent 125, and the preparation of the PCR reaction solution is completed.

[0066] (4) Microcavity injection and oil sealing: After the elution process is completed, tape is used to seal the other storage chambers except the mineral oil storage chamber 114. By continuously injecting the oil phase into the mineral oil storage chamber 114 and applying pressure, the oil phase pushes the water phase into the lower microcavity injection port 301 in an orderly manner through the microcavity detection layer injection port connection hole 105. The reagent filling and oil sealing steps of the microporous reaction chamber 303 are completed through the flow channel 302, and the excess reagent is discharged from the chip through the sample outlet 304 and the microcavity detection layer sample outlet connection hole 106.

[0067] (5) Amplification detection: The nucleic acid extraction, purification and digital detection chip is placed in an in-situ PCR instrument and the amplification program is run. When the amplification program is completed and the device cools to room temperature, the chip is placed under a fluorescence microscope to observe the amplification results.

[0068] (6) Data analysis: Use Image to capture fluorescence images ( Figure 6 ) is analyzed by counting the number of positive microwells in each chip and combining it with the Poisson distribution principle to obtain the starting concentration of the target nucleic acid in the sample to be tested, thereby achieving qualitative and quantitative detection of pathogens.

Claims

1. A multilayer microfluidic chip integrating nucleic acid extraction and purification with digital detection, characterized in that: The multilayer microfluidic chip comprises, from top to bottom, a PDMS-based nucleic acid extraction and purification layer (100), a glass substrate (200) and a silicon-based microcavity digital PCR detection layer (300); wherein the PDMS-based nucleic acid extraction and purification layer (100) is composed of a liquid storage chamber (110), a bottom connecting pipe (101), a microcolumn array (102), a capillary burst valve (104), a microcavity detection layer sample inlet connection hole (105) and a microcavity detection layer sample outlet connection hole (106); the liquid storage chamber (110) comprises a lysate storage chamber (111), a first washing liquid storage chamber (112), a second washing liquid storage chamber (113), a mineral oil storage chamber (114) and an eluent storage chamber (115) which are sequentially connected via the bottom connecting pipe (101); the microcolumn array (102) is used for the mineral oil storage chamber (114) and the eluent storage chamber (115); 115); the capillary burst valve (104) is used to prevent the liquid in the eluent storage chamber (115) from spontaneously entering the microcavity detection layer sample inlet connection hole (105) during the nucleic acid purification process; the glass substrate (200) is provided with openings corresponding to the microcavity detection layer sample inlet connection hole (105) and the microcavity detection layer sample outlet connection hole (106); the silicon-based microcavity digital PCR detection layer (300) comprises A microcavity injection port (301), a flow channel (302), a microporous reaction chamber (303) and a sample outlet (304); the microcavity injection port (301) is connected to a microcavity detection layer injection port connection hole (105), and the sample outlet (304) is connected to a microcavity detection layer sample outlet connection hole (106); the microporous reaction chamber (303) is connected to the microcavity injection port (301) and the sample outlet (304) via the flow channel (302).

2. The multilayer microfluidic chip according to claim 1, characterized in that: The lysing liquid storage chamber (111), the first washing liquid storage chamber (112), and the second washing liquid storage chamber (113) utilize the water-in-oil principle to achieve non-interference between the lysing liquid, the first washing liquid, and the second washing liquid.

3. The multilayer microfluidic chip according to claim 1, characterized in that: The eluent storage chamber (115) is provided with an eluent injection hole (103).

4. A method for using the multilayer microfluidic chip integrating nucleic acid extraction and purification with digital detection as claimed in claim 1, comprising the following steps: (1) firstly, pre-fill the aqueous elution reagent into the elution liquid storage chamber (115), then inject mineral oil into the lysis liquid storage chamber (111), and tilt the chip to allow the mineral oil to flow through the first washing liquid storage chamber (112), the second washing liquid storage chamber (113), and the mineral oil storage chamber (114) through the bottom connecting pipe (101) and fill them; then, inject the nucleic acid extraction and purification reagent into the corresponding storage chamber, and each reagent is separated in an independent chamber; (2) During the detection process, the sample is added to the lysis solution storage chamber (111), the cells or viruses in the sample are lysed, and the released nucleic acid molecules are adsorbed by the nucleic acid extraction magnetic beads (131), and then the nucleic acid extraction magnetic beads (131) are dragged into the subsequent reaction chamber to complete the washing and elution process to obtain the PCR reaction solution; (3) The other liquid storage chambers except the mineral oil liquid storage chamber (114) are sealed with adhesive tape, and the oil phase is continuously injected into the mineral oil liquid storage chamber (114) to apply pressure, so that the oil phase pushes the water phase into the silicon-based microcavity digital PCR detection layer (300) in an orderly manner through the microcavity detection layer injection port connection hole (105); finally, the chip is placed in an in-situ PCR instrument and the relevant program is set, and the detection result is observed under a fluorescence microscope after the reaction is completed.

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